大豆耐低磷转录因子GmPTF1与GmPHR1功能分析

发布时间：2018-07-21 19:10

【摘要】：磷是植物生长发育必需营养元素，但土壤有效磷缺乏严重影响了作物产量提高与品质改良。发掘利用植物耐低磷相关基因，创制磷高效作物新品种是解决上述问题最佳途径。本研究以课题组前期克隆的转录因子基因GmPTF1与GmPHR1为对象，分析转录因子的激活活性位点、细胞内定位、品种间的序列与表达差异及其在转基因拟南芥中的生物学功能，旨在获得具有自主知识产权的耐低磷基因，为重要农作物转基因育种提供功能基因。主要结果如下： 1.生物信息学比对分析发现，GmPTF1全长8275bp，由6个内含子、7个外显子组成。GmPHR1全长2751bp，包含5个内含子与6个外显子。GmPTF1和GmPHR1在基因组中各有2个拷贝，均位于3号、19号染色体。 2.不同大豆品种基因序列分析发现，GmPTF1的ORF序列在中黄15（耐低磷）与牛毛黄（不耐低磷）间没有差异。GmPHR1的ORF序列在中黄15、冀豆11（耐低磷）与牛毛黄间存在1个碱基突变，导致编码蛋白在第225位出现1个氨基酸的改变。 3.实时定量PCR分析基因表达发现，低磷处理下，GmPTF1主要在大豆根系表达，从低磷处理开始直至56d，，GmPTF1在中黄15中的表达量高于牛毛黄。GmPHR1主要在茎杆表达，在冀豆11中被快速诱导，0.5h出现第1个表达高峰，随后稍有下降（6h），随即又被快速诱导，12-24h出现第2个表达高峰；在牛毛黄中被缓慢诱导，6h出现高峰，随即表达量快速下降，48h达到最低；且GmPHR1在冀豆11的表达量从低磷处理0.5-48h始终高于牛毛黄。 4.酵母单杂交分析基因编码蛋白转录激活活性发现，GmPTF1转录激活活性位点位于蛋白N端和C端，GmPHR1转录激活活性位点位于蛋白C端。亚细胞定位与荧光显微观察转GmPTF1与GmPHR1拟南芥根系发现，GmPTF1与GmPHR1编码蛋白均定位于细胞核。 5.转基因拟南芥与野生型低磷处理试验发现，转GmPTF1超表达植株的根冠比高于野生型与RNAi植株，RNAi植株最大根长低于野生型与超表达植株，GmPTF1具有促进低磷条件下转基因拟南芥根系生长的作用。转GmPHR1超表达植株的地上部干重、根系干重、植株总干重、根冠比、最大根长均优于野生型与RNAi植株，而RNAi植株根系干重、植株总干重低于野生型，GmPHR1具有提高低磷条件下转基因拟南芥磷素利用效率的功能。
[Abstract]:Phosphorus is an essential nutrient for plant growth and development, but the lack of available phosphorus in soil seriously affects crop yield and quality improvement. The best way to solve the above problems is to explore and utilize plant low phosphorus tolerance genes and to create new varieties of high phosphorus efficiency crops. In this study, the transcriptional factor genes GmPTF1 and GmPHR1, which were cloned earlier in the study group, were used to analyze the activation sites, intracellular localization, sequence and expression differences of transcription factors and their biological functions in transgenic Arabidopsis thaliana (Arabidopsis thaliana). The aim of this paper is to obtain low phosphorus tolerance genes with independent intellectual property rights and to provide functional genes for transgenic breeding of important crops. The main results are as follows: 1. Bioinformatics analysis showed that GmPTF1 was 8275 BP in length, consisting of 6 introns and 7 exons. GmPHR1 was composed of 5 introns and 6 exons. GmPTF1 and GmPHR1 had 2 copies each in the genome, all located on chromosome 3 and 19. The ORF sequence of GmPTF1 was not different between Zhonghuang 15 (low phosphorus tolerance) and Bovine hair Yellow (low P tolerance). The ORF sequence of GmPHR1 was 1 base mutation between Zhonghuang 15, Jidou 11 (low phosphorus tolerance) and Bovine hair Yellow. The result is that the encoded protein has a change of 1 amino acid at 225th position. 3. 3. Real-time quantitative PCR analysis showed that GmPTF1 was mainly expressed in soybean root system under low phosphorus treatment, and the expression of GmPTF1 in Zhonghuang 15 was higher than that in Bovine hair Yellow. GmPHR1 was mainly expressed in stem from low phosphorus treatment until 56d. The first peak of expression appeared at 0.5 h, then decreased slightly (6 h), and then the second peak appeared in 12 to 24 h after rapid induction, and the peak appeared at 6 h in cattle hair yellow. The expression of GmPHR1 in Jidou11 was decreased rapidly at 48h, and the expression of GmPHR1 in Jidou11 was always higher than that of Bovine hair yellow from 0.5-48h of low phosphorus treatment. The transcriptional activation activity of GmPTF1 was found to be located at the N-terminal and C-terminal of GmPHR1. Subcellular localization and fluorescence microscopical observation of GmPTF1 and GmPHR1 in Arabidopsis thaliana root system it was found that both GmPTF1 and GmPHR1 encoded proteins were located in the nucleus. Transgenic Arabidopsis thaliana and wild type low phosphorus treatment showed that The root / shoot ratio of transgenic GmPTF1 superexpressed plants was higher than that of wild type and RNAi plants. The maximum root length of transgenic plants was lower than that of wild type and overexpression plants. GmPTF1 could promote the root growth of transgenic Arabidopsis thaliana under low phosphorus. The aboveground dry weight, root dry weight, total plant dry weight, root to shoot ratio, maximum root length of GmPHR1 overexpressed plants were superior to those of wild type and RNAi plants, while the root dry weight of RNAi plants was better than that of wild type and RNAi plants. The total dry weight of plants was lower than that of wild type GmPHR1, which could improve the phosphorus utilization efficiency of transgenic Arabidopsis thaliana under low phosphorus.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S565.1

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